|Year : 2014 | Volume
| Issue : 1 | Page : 73-81
Insights from computational analysis of full-length β-ketoacyl-[ACP] synthase-II cDNA isolated from American and African oil palms
Subhash J Bhore1, Thye S Cha2, Kassim Amelia1, Farida H Shah3
1 Department of Molecular Biology, Melaka Institute of Biotechnology, Lot 7, Melaka International Trade Centre City, 75450 Ayer Keroh, Melaka; Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Bedong Semeling Road, Bedong, 08100, Kedah, Malaysia
2 School of Bioscience and Biotechnology, Faculty of Science and Technology, National University of Malaysia (UKM), 43600 Bangi, Selangor; Department of Biological Sciences, Faculty of Science and Technology, Universiti Malaysia Terengganu (UMT), 21030 Kuala Terengganu, Malaysia
3 Department of Molecular Biology, Melaka Institute of Biotechnology, Lot 7, Melaka International Trade Centre City, 75450 Ayer Keroh, Melaka; School of Bioscience and Biotechnology, Faculty of Science and Technology, National University of Malaysia (UKM), 43600 Bangi, Selangor, Malaysia
|Date of Web Publication||18-Feb-2014|
Subhash J Bhore
Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Bedong Semeling Road, Semeling 08100, Kedah
Source of Support: Authors are grateful to the Ministry of Science,
Technology and Innovation (MOSTI) of Malaysian Government for
research funding [Grant Code: IRPA: 01-02-02-0014/PR0015/07-07]., Conflict of Interest: None
| Abstract|| |
Background: Palm oil derived from fruits (mesocarp) of African oil palm (Elaeis guineensis Jacq. Tenera) and American oil palm (E. oleifera) is important for food industry. Due to high yield, Elaeis guineensis (Tenera) is cultivated on commercial scale, though its oil contains high (~54%) level of saturated fatty acids. The rate-limiting activity of beta-ketoacyl-[ACP] synthase-II (KAS-II) is considered mainly responsible for the high (44%) level of palmitic acid (C 16:0 ) in the oil obtained from E. guineensis. Objective: The objective of this study was to annotate KAS-II cDNA isolated from American and African oil palms. Materials and Methods: The full-length E. oleifera KAS-II (EoKAS-II) cDNA clone was isolated using random method of gene isolation. Whereas, the E. guineensis KAS-II (EgTKAS-II) cDNA was isolated using reverse transcriptase polymerase chain reaction (RT-PCR) technique; and missing ends were obtained by employing 5'and 3' RACE technique. Results: The results show that EoKAS-II and EgTKAS-II open reading frames (ORFs) are of 1689 and 1721 bp in length, respectively. Further analysis of the both EoKAS-II and EgTKAS-II predicted protein illustrates that they contains conserved domains for 'KAS-I and II', 'elongating' condensing enzymes, 'condensing enzymes super-family', and '3-oxoacyl-[ACP] synthase II'. The predicted protein sequences shows 95% similarity with each other. Consecutively, the three active sites (Cys, His, and His) were identified in both proteins. However, difference in positions of two active Histidine (His) residues was noticed. Conclusion: These insights may serve as the foundation in understanding the variable activity of KAS-II in American and African oil palms; and cDNA clones could be useful in the genetic engineering of oil palms.
Keywords: African oil palm, American oil palm, cooking oil, edible oil, Elaeis oleifera, Elaeis guineensis, fatty acids, oil palm, palm oil
|How to cite this article:|
Bhore SJ, Cha TS, Amelia K, Shah FH. Insights from computational analysis of full-length β-ketoacyl-[ACP] synthase-II cDNA isolated from American and African oil palms. J Nat Sc Biol Med 2014;5:73-81
|How to cite this URL:|
Bhore SJ, Cha TS, Amelia K, Shah FH. Insights from computational analysis of full-length β-ketoacyl-[ACP] synthase-II cDNA isolated from American and African oil palms. J Nat Sc Biol Med [serial online] 2014 [cited 2017 Sep 22];5:73-81. Available from: http://www.jnsbm.org/text.asp?2014/5/1/73/127292
| Introduction|| |
Oil palm is an important and a major source of edible oil. The oil palms belong to the family Arecaceae which contains 202 genera and about 2600 species distributed in tropical, subtropical, and warm temperate climates. Genus Elaeis contains only two oil palm species namely, Elaeis guineensis Jacq. and E. oleifera. The E. guineensis and E. oleifera are referred as African oil palm and American oil palm, respectively. The E. guineensis Jacq. contains three fruit forms (also called as verities) namely, 'Dura', 'Pisifera', and 'Tenera' that are distinguished based on the shell thickness of fruits. , Elaeis guineensis Jacq. 'Tenera' a hybrid of 'Dura' (♀) and 'Pisifera' (♂) is highly preferred for the commercial cultivation because of its high oil-yielding fruits. 
In tropical countries like Malaysia and Indonesia, oil palm is a major agricultural crop and both countries are major producers and exporters of the palm oil. Elaeis oleifera is not extensively cultivated for the palm oil due to low oil-yielding fruits. , However, palm oil obtained from E. oleifera fruit mesocarp is far superior to palm oil obtained from commercially cultivated E. guineensis Jacq. 'Tenera' fruit mesocarp. 
The palm oil obtained from E. guineensis (Tenera) and E. oleifera contains about 44% and 25% palmitic acid (C 16:0 ), respectively.  The level of the C 16:0 in palm oil is dependent on the palmitoyl-acyl carrier protein [ACP] thioesterase (PATE) (EC: 188.8.131.52) and beta-ketoacyl-[ACP] synthase-II (KAS-II) (EC: 184.108.40.206) enzymes efficiency. , However, with advanced tools and techniques available for the plant genetic transformation, fatty acid biosynthesis pathway can be manipulated genetically to improve palm oil quality or to add value in it. The level of C 16:0 content in E. guineensis oil can be reduced by PATE gene silencing and over expression of the KAS-II gene in a mesocarp tissue-specific manner.  Researchers are working hard to transform oil palm for PATE gene silencing and successful oil palm transformation is reported elsewhere. ,,
In addition to PATE, KAS-II gene is an important gene target for the genetic modification of oil palm to minimize the C 16:0 content. The KAS-II enzyme catalyzes the elongation of C 16:0 -ACP to C 18:0 -ACP in plastidial fatty acid biosynthesis pathway. The biological activity of KAS-II is considered as rate limiting in commercially cultivated E. guineensis compared to its activity in E. oleifera., In order to understand the gene expression patterns in E. oleifera fruit mesocarp tissue, expressed sequence tags (ESTs) generation project was initiated at Melaka Institute of Biotechnology, Melaka; and until now, 3,205 ESTs are generated from 17-week-old E. oleifera fruit-mesocarp tissue cDNA library (unpublished work). The EoKAS-II is one of the isolated cDNA (EST) clones.
The computational annotation of EoKAS-II and EgTKAS-II and its comparative analysis could be useful in elucidating similarities and differences between EoKAS-II and EgTKAS-II. This could also give some insights in in-depth understanding of fatty acid biosynthesis pathway in both Elaeis species. The EoKAS-II and EgTKAS-II gene's cDNA nucleotide sequences, deduced proteins sequences as well as annotations are reported in this paper.
| Materials and Methods|| |
Bacto-Agar, Bacto-Trypton, and Yeast extracts were purchased from DIFCO Laboratories (Etroit MI, USA). Chloroform, EDTA, ethanol, iso-amyl alcohol and sodium chloride were from BDH Laboratory, England. The 100 bp DNA ladder, λ DNA Hind III DNA marker, the deoxy-nucleotide mix (dNTPs), other PCR components and Wizard® Plus SV Minipreps DNA purification system (kit) were purchased from Promega Corporation, USA. Restriction enzymes used were procured from New England BioLabs® Inc. The CloneMiner TM cDNA Library Construction Kit was procured from Invitrogen Corporation; and all other chemicals except those are mentioned above were procured from the Sigma-Aldrich Corporation (St. Louis, MO), USA.
EgTKAS-II and EoKAS-II cDNA isolation
Elaeis oleifera , 17-week-old [weeks after anthesis (WAA)] fruit mesocarp tissue cDNA library constructed using the 'CloneMiner cDNA library construction kit' for ESTs generation (unpublished work) was used to isolate EoKAS-II. This EoKAS-II cDNA clone was isolated by random method of cDNA isolation.  Whereas, the reverse transcriptase polymerase chain reaction (RT-PCR) technique was used to isolate EgTKAS-II cDNA from E. guineensis. The forward (5'- AGAATTGCCGGAGAGATCAAGTC-3') and reverse (5'- GATGAGTTATGACCACCAAACC-3') primers were designed based on the conserved regions of KAS-II gene from Cuphea wrightii (U67317) and Perilla frutescene (AF026149). The RNA samples extracted from 17-week-old (WAA) fruit mesocarp-tissues of E. guineensis were used. The PCR for synthesis and amplification of EgTKAS-II cDNA was completed using: hot start at 94°C for 5 min, followed by 35 cycles of 33 sec at 94°C, 1 min at 49°C, 1.30 min at 72°C and a final extension at 72°C for 5 minutes. The 5' RACE and 3'-RACE techniques were used to isolate the missing 5' and 3'end of the EgTKAS-II gene cDNA, respectively. The PCR cloning vector, pGEM® -T Easy was used for the cloning of the PCR products.
Plasmid DNA purification and cDNA sequencing
Separately, the Escherichia More Details coli strain DH5α cells harboring EoKAS-II and EgTKAS-II cDNA were cultivated in 10-ml LB medium in dark at 37°C, 160 rpm for 16-18 h. The cultures of E. coli cells harboring plasmid DNA with EoKAS-II and EgTKAS-II cDNA were supplemented with Kanamycin (50 μg/ml) and Ampicillin (50 μg/ml), respectively. The E. coli cells were harvested from broth and plasmid DNA was isolated by using a commercial kit, Wizard® Plus SV Minipreps DNA purification system.
For EoKAS-II cDNA, sequencing reactions were carried out for both strands using M13 (Forward); 5'-GTAAAACGACGGCCAG-3' and M13 (Reverse); 5'- GGATAACAATTTCACACAGG-3' primers. Whereas, for EgTKAS-II cDNA, sequencing reactions were carried out for both strands using T7 and SP6 universal primers available in PCR cloning vector, pGEM® -T Easy.
Computational analysis of nucleotide and protein sequence
The sequences of cDNAs were edited manually to eliminate vector and or adaptor sequences from 5' and 3' ends. Blastn and Blastx programs were used to confirm the identity of the both isolated cDNA. The general features of the EoKAS-II and EgTKAS-II cDNA, conserved domains, and multiple sequence alignment of protein sequences was carried out as described by Bhore et al.  The active sites (residues) of EoKAS-II and EgTKAS-II proteins were identified based on active sites of E. coli KAS protein (1FJ8_A) described by Price et al. 
| Results|| |
A full-length EoKAS-II cDNA clone was isolated from 17-week-old fruit-mesocarp tissue cDNA library. Its nucleotide sequence, deduced protein sequence, and other features are shown in [Figure 1]. The nucleotide sequence, sequence isolated using 5' and 3' RACE technique as well as the deduced protein sequence and other features of EgTKAS-II cDNA are depicted in [Figure 2]. The nucleotide sequence of EoKAS-II and EgTKAS-II gene cDNA has been submitted to GenBank/DDBJ/EMBL under accession numbers FJ940767 and AF220453, respectively.
|Figure 1: Nucleotide and deduced amino acid sequence of E. oleifera ƒÒ-ketoacyl-[ACP] synthase II (EoKAS-II) cDNA clone. An open reading frame and non-coding regions are shown in capital and small letters, respectively. The deduced amino acid sequence is given below the nucleotide sequence, which is numbered at the both ends of each sequence line. The open reading frame encodes for a protein of 562 amino acid residues. Amino acid residues are numbered beginning with the initial Methionine (m) till last Lysine (k) residue. Initiation and termination codons are shown in bold. The 'ƒÒ-ketoacyl-[ACP] synthase (KAS) (type I and II)', 'Elongating' condensing enzymes and 'Condensing enzymes super-family|
conserved domain (multi-domain) residues (170 to 543) are highlighted in green color. Residues (1 to 562) underlined with pink color line are part of the '3-oxoacyl-[ACP] synthase II' domain (region ame="PLN02787"). *represent the termination codon. This cDNA clone was isolated by random method of gene isolation from E. oleifera 17-week-old fruit-mesocarp tissue cDNA library
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|Figure 2: Nucleotide and deduced amino acid sequences of E. guineensis (Tenera) ƒÒ-ketoacyl-[ACP] synthase II (EgTKAS-II) cDNA clone. An open reading frame and noncoding regions are shown in capital and small letters, respectively. Nucleotides shown in bold brown color represent the location of the forward primer (3'-RACE FP) used in 3'-RACE and reverse primer (5'-RACE RP) used in 5'-RACE. Arrows indicate the 5'to 3' direction of the primer. Nucleotides shown in brown color represent the overlapping regions with cDNA fragments isolated using 5' and 3'-RACE. The deduced amino acid sequence is given below the nucleotide sequence, which is numbered at the both ends of each sequence line. The open reading frame encodes for a protein of 573 amino acid residues. Amino acid residues are numbered beginning with the initial Methionine (m) till last Cysteine (c) residue. Initiation and termination codons are shown in green and red color, respectively. The 'ƒÒ-ketoacyl-[ACP] synthase (KAS) (type I and II)', 'Elongating' condensing enzymes and 'Condensing enzymes super-family conserved domain (multi-domain) residues (174 to 546)|
are highlighted in green color. Residues (1 to 563) underlined with pink color line are part of the '3-oxoacyl-[ACP] synthase II' domain (region name="PLN02787"). *represent the termination codon. This cDNA was isolated from E. guineensis 17-week-old fruit-mesocarp tissue by using RT-PCR, and 5' and 3' RACE techniques.
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Analysis of cDNAs general features [Table 1] revealed that guanine plus cytosine content (G + C %) in both EoKAS-II and EgTKAS-II gene cDNA was 49%. The alignment of the deduced proteins showing similarities and differences between EgTKAS-II and EoKAS-II is shown in [Figure 3]. Consecutively, the multiple sequence alignment was completed, and the three active sites were identified as shown in [Figure 4]a [for complete multiple sequence alignment, see [Figure 5]. The phylogenetic tree was constructed to find out phylogenetic relationship among compared KAS-II protein sequences; and the radial phylogenetic tree is shown in [Figure 4]b.
|Figure 3: Comparison between EoKAS-II and EgTKAS-II amino acid sequence depicts the similarity and difference. Amino acid sequences are numbered at the end of each sequence row. (*), (:) and (.) indicate identical residues, residues with conserved strong groups and residues with conserved weak groups, respectively. EgTKAS-II and EoKAS-II represents the Elaeis guineensis Jacq. (Tenera) and Elaeis oleifera ƒÒ-ketoacyl-[ACP] synthase II amino acid sequence, respectively|
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|Figure 4: (a) Multiple sequence alignment of EoKAS-II and EgTKAS-II amino acid sequences with their selected counterparts from other organism. Protein sequences were aligned using the CLUSTALW alignment algorithm. The three active sites (residues) of EoKAS-II and EgTKAS-II proteins corresponding to E. coli KAS, Cys163, His298, and His333 are highlighted in pink color; the numbers at top indicate the residue ruler for whole alignment. GenBank accession numbers are as follows: Eo, FJ940767; EgT, AF220453; Jc, DQ987700; Gh, HM236494; Rc, EQ973802; Ah, EU823327; Cw, U67317; Pf, AF026149; Gm, AY907523; Ha, DQ835562; Bn, AF244520; Cr, DS496157; Ec, 1FJ8_A. Abbreviations: Eo, Elaeis oleifera; EgT, Elaeis guineensis (Tenera); Jc, Jatropha curcas; Gh, Gossypium hirsutum; Rc, Ricinus communis; Ah, Arachis hypogaea; Cw, Cuphea wrightii; Pf, Perilla frutescens; Gm, Glycine max; Ha, Helianthus annuus; Bn, Brassica napus; Cr, Chlamydomonas reinhardtii; Ec, Escherichia coli. This alignment is produced by CLUSTAL W multiple sequence alignment program. (b) Radial phylogenetic tree of amino acid sequence|
relationship among the proteins aligned in (a). The phylogenetic distance scale at bottom is proportional to the divergence between the sequences.
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|Figure 5: Multiple sequence alignment of EoKAS-II and EgTKAS-II proteins with their selected counterparts from other organism. Protein sequences were aligned using the CLUSTALW alignment algorithm. Identical and conserved residues are shaded black and grey, respectively; the numbers at top indicate the residue ruler for whole alignment. GenBank accession numbers are as follows: Eo, FJ940767; EgT, AF220453; Jc,|
DQ987700; Gh, HM236494; Rc, EQ973802; Ah, EU823327; Cw, U67317; Pf, AF026149; Gm, AY907523; Ha, DQ835562; Bn, AF244520; Cr, DS496157; Ec, 1FJ8_A. Abbreviations: Eo, Elaeis oleifera; EgT, Elaeis guineensis (Tenera); Jc, Jatropha curcas; Gh, Gossypium hirsutum; Rc,Ricinus communis; Ah, Arachis hypogaea; Cw, Cuphea wrightii; Pf, Perilla frutescens; Gm, Glycine max; Ha, Helianthus annuus; Bn, Brassica napus; r, Chlamydomonas reinhardtii; Ec, Escherichia coli. This alignment is roduced by CLUSTAL W multiple sequence alignment program
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|Table 1: General features of American (Eo) and African (EgT) oil palms full-length ß-ketoacyl-|
[ACP] synthase II (KAS-II) gene's cDNA sequence
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| Discussion|| |
Oil palm is an important oil producing crop and nutritional quality of its oil can be improved by using tools available for plant genetic engineering. However, in order to make a strategy for genetic engineering of socio-economically important crop, oil palm; understanding of its key genes involved in fatty acid biosynthesis pathway is necessary. Therefore, several key genes that encodes for enzymes involved in fatty acid biosynthesis pathway were isolated in our laboratory. In this study, we determined the full-length cDNA sequence of EoKAS-II and EgTKAS-II which are known for catalyzing the elongation of C 16:0 fatty acid-[ACP] to C 18:0 fatty acid-[ACP] in plastids. 
We performed sequencing for both positive and negative strands of both EoKAS-II and EgTKAS-II cDNA clones. The complete EoKAS-II and EgTKAS-II cDNA sequence was 2138 and 2011 bp in length, respectively [Figure 1] and [Figure 2]. The EoKAS-II and EgTKAS-II cDNA's open reading frame (ORF) encodes for 562 and 573 amino acids, respectively. Both, EoKAS-II and EgTKAS-II ORF contains conserved domains for 'KAS-I and II', which is responsible for the elongation steps in fatty acid biosynthesis process.  The ORFs of EoKAS-II and EgTKAS-II gene also contain 'Condensing enzymes super-family'  and '3-oxoacyl-[ACP] synthase II'  conserved domains.
From protein BLAST hits analysis, we observed that EoKAS-II and EgTKAS-II proteins are 95% identical with each other [Figure 3]. However, Brassica napus KAS-II (AF244520) protein was the closest to EoKAS-II and EgTKAS-II proteins. It was reflected in multiple sequence alignment of the proteins [see [Figure 5] as well as in the phylogenetic tree of the KAS-II proteins [Figure 4]b. The KAS sequence from Chlamydomonas reinhardtii (DS496157)  and E. coli (1FJ8_A)  showed maximum phylogenetic distance with the EoKAS-II and EgTKAS-II proteins [Figure 4]b. The phylogenetic distance of EoKAS-II and EgTKAS-II with their counterparts from C. reinhardtii and E. coli is in line with the evolution of living organisms. However, three active sites (Cys-His-His) , were fully conserved in EoKAS-II (Cys316, His453, and His489), EgTKAS-II (Cys316, His456, and His492) and their counterpart proteins from other species, except Gossypium hirsutum KAS-II (HM236494) protein in which one active residue (His) was missing [Figure 4]a.
Both, EgTKAS-II and EoKAS-II gene cDNA clones were isolated from 17-week-old fruit-mesocarp tissue; and this indicated that EgTKAS-II and EoKAS-II genes were expressed in developing fruit mesocarp tissue of both Elaeis species. However, it is also expressed in leaf, roots (unpublished data) and flower tissues, and embryogenic callus.  It also suggests that EgTKAS-II and EoKAS-II genes are not tissue-specific in their expression.
Palm oil derived from fruit mesocarp tissue of E. oleifera contains about 68.6% oleic acid (C 18:1 ) and 25% C 16:0 . However, C 18:1 and C 16:0 content in the palm oil obtained from fruit mesocarp of commercially cultivated E. guineensis Jacq. Tenera is 32.5% and 44%, respectively.  The total content of saturated fatty-acids in palm oil from commercially cultivated E. guineensis is 52%; and it is significantly high in comparison to other vegetable oils and fats [Figure 6].  The PATE enzyme is known to have C 16:0- ACP substrate specificity; ,, and its high efficacy is responsible in part for high (44%) content of C 16:0 in palm oil derived from E. guineensis. Hence, the nutritional quality of the palm oil obtained from commercially cultivated E. guineensis can be improved by minimizing the C 16:0 content. This can be done by silencing the expression of PATE gene and overexpression of the EgTKAS-II gene. Therefore, the isolated EgTKAS-II cDNA clone will be useful in genetic transformation of oil palms in general and commercially cultivated E. guineensis in particular. The EoKAS-II cDNA clone could be useful in genetic transformation experiments as a reference so that its efficiency can be compared with efficiency of EgTKAS-II.
|Figure 6: Bar diagram showing saturated and unsaturated fatty acids (%) in edible vegetable oils and fats. Palm oil represented is from commercially cultivated oil palm, Elaeis guineensis Jacq. Tenera; Svs,|
solid vegetable shortening; Pki, Palm Kernel oil.
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As a part of oil palm molecular biology project, previously, we have isolated mesocarp tissue-specific  and kernel tissue-specific  cDNA clones in order to study the genes expressed during oil palm fruit development and to have tissue-specific gene promoters for oil palm genetic transformation. For the PATE gene silencing, various novel expression cassettes were constructed  and genetic transformation work was initiated in our laboratory. , However, EgTKAS-II gene expression cassette for it's over expression and PATE gene-silencing expression cassettes can be incorporated in a single vector to utilize them in genetic transformation of commercially cultivated E. guineensis. It may help to save the time in producing genetically engineered oil palm (E. guineensis) which can produce more stearic acid (C 18:0 ) and or C 18:1 at the expense of C 16:0 .
The use of EgTKAS-II and EoKAS-II gene promoters or oil palm fruit mesocarp tissue-specific gene promoters for their overexpression could be an advantage. Due to commercial importance of oil palm, Malaysian Palm Oil Board has completed oil palms (E. oleifera and E. guineensis Jacq. including the Pisifera and Dura) genome sequencing; however, the data is not available in the public domain.  The Agrobacterium tumefaciens-mediated  or biolistic mediated  mode of genetic transformation for oil palm can be used once the expression cassettes are ready for the over expression of KAS-II. However, the alternative of chloroplast transformation can also be considered for the genetic transformation of oil palm. ,
| Conclusions|| |
In conclusion, we determined the full-length cDNA sequence of the KAS-II gene from American and African oil palms. We showed three active sites (catalytic triad) and the conserved domains in both EoKAS-II and EgTKAS-II deduced protein. These two cDNA clones are important for the genetic engineering of fatty acid biosynthesis pathway in oil palm to improve nutritional quality of the palm oil. However, the biochemical characterization of the EoKAS-II and EgTKAS-II proteins is not done yet and need further study in this line to understand the difference in the efficacy of this enzyme in two Elaeis species. Furthermore, an investigation of the structural differences between EoKAS-II and EgTKAS-II protein is warranted.
| Acknowledgments|| |
Authors are grateful to the Ministry of Science, Technology and Innovation (MOSTI) of Malaysian Government for research funding [Grant Code: IRPA: 01-02-02-0014/PR0015/07-07], and to the United Plantation Berhad, Perak, Malaysia (now merged with Sime Durby) for supplying fruit samples used in this study.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]